S120: ACUTE MYELOID LEUKEMIA REPRESENTS A FERROPTOSIS-SENSITIVE CANCER ENTITY RAISING THE POSSIBILITY FOR NOVEL TARGETING STRATEGIES
Authors: A. Narr, H. Alborzinia, E. Donato, F. Vogel, T. Boch, A.-M. Leppä, A. Waclawiczek, S. Renders, A. Schulze, A. Trumpp
Journal: HemaSphere (2022)

Background: Despite advances in the treatment of Acute Myeloid Leukemia (AML), the 5-year patient survival rate remains poor with 15-20%. The majority of patients develop recurrence partly due to resistance mechanisms to classical cell death programs. Exploiting new forms of cell death therefore may allow novel therapeutic options to limit survival or occurrence of resistant clones. Ferroptosis has been identified as non-apoptotic and iron-dependent form of cell death, characterized by an excessive ROS-induced peroxidation of cell membranes. Recently it has been implicated with a higher sensitivity in cancer stem cells and drug-resistant cancer cells, potentially making ferroptosis-inducing (FIN) therapies a promising option. Aims: We aim to elucidate whether AML represents a ferroptosis-sensitive cancer entity and is susceptive for potential FIN-therapies. Methods: IC50 values of ferroptosis inducers were determined and used in combination with the TCGA-LAML and a pan-cancer CRISPR screen dataset (CERES) to describe ferroptosis sensitivity of AML. A drug screen with known ROS-inducers was performed in 9 AML cell lines to identify ferroptosis induction. To mechanistically characterize ferroptotic cell death by the identified drug on the genetic and metabolic level, we performed SLAM-RNA-, RNA-Sequencing, Mass Spectrometry and Metabolite Tracing (Cystine/Ser/Gln) in HL-60 cells in vitro and in vivo. Genetic (CRISPR-Cas9-KO) and chemical inhibition were used to characterize the role of identified hits and pathways, aiming to design new synergistic combination therapies. Results: We find that AML belongs to the most dependent cancer entities for various ferroptosis-associated genes and represents a ferroptosis-sensitive cancer entity. Furthermore, a 12-gene-ferroptosis-signature allows us to predict the Overall Survival of AML patients and shall help to identify a potential cohort for FIN-therapies. By screening known ROS-inducing drugs for ferroptosis induction, we identify one drug capable of inducing lipid peroxidation and subsequently ferroptosis in multiple AML and other tumor cell lines. Notably, in detail characterization of the drug’s mechanism on the genetic and metabolic level reveal the activity of the iron-metabolism gene HMOX-1 as essential for ferroptosis induction. Mechanistically, we further find that this drug directly inhibits GPX-4, one of the major ferroptosis-suppressive regulators, and facilitates its degradation. Furthermore, we demonstrate that drug treatment results in an upregulation of different metabolic pathways involved in glutathione (GSH) synthesis; namely (1) SLC7A11-mediated Cystine-uptake, (2) transsulfuration pathway as well as (3) glutaminolysis. Genetical and chemical inhibition of these pathways together with drug treatment in in vitro as well as in vivo studies shows strong synergistic effects marking them as new targetable vulnerabilities in AML. Using these insights of the drug’s mechanism of action, we design a synergistic combination therapy and demonstrate its tumor-eradicating capacity in AML cell lines (in vitro & in vivo) and in primary de novo as well as relapse AML patient samples (ex vivo). Summary/Conclusion: These findings classify AML as ferroptosis sensitive and reveal the capacity of this drug to induce ferroptosis. Detailed characterization of the drug’s mechanism on the genetic and metabolic level allows us to design an effective combination therapy exploiting newly identified vulnerabilities in AML. These results provide a rational basis for the additional pre-clinical and subsequent clinical evaluation of FIN-therapies in AML patients.